Assessment of anionic siRNA lipoplexes prepared via modified ethanol injection for tumor cell delivery

Our previous study introduced a modified ethanol injection (MEI) method for preparing positively charged small interfering RNA (siRNA) lipoplexes by mixing a lipid-ethanol solution of cationic lipid, 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphoethanolamine (DOPE), and poly(ethylene glycol) cholesteryl ether (PEG-Chol) with siRNA in phosphate-buffered saline (PBS). This method was adapted in the current study to develop a two-step MEI process for creating anionic siRNA lipoplexes. First, a lipid-ethanol solution comprising one of four cationic lipids [1,2-dioleoyl-3-trimethylammonium-propane methyl sulfate salt (DOTAP), dimethyldioctadecylammonium bromide (DDAB), <i>N</i>-hexadecyl-<i>N</i>, <i>N</i>-dimethylhexadecan-1-aminium bromide (DC-1-16), or 11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-<i>N</i>,<i>N</i>,<i>N</i>-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12)], DOPE and PEG-Chol was combined with siRNA in PBS. Next, a lipid-ethanol solution of cholesteryl hemisuccinate (CHS) and DOPE was added. The gene-silencing activity of anionic siRNA lipoplexes was evaluated in human breast cancer (MCF-7) and human cervical carcinoma (HeLa) cells. Additionally, their interactions with erythrocytes were investigated. Regardless of the cationic lipid used, adding the CHS and DOPE-ethanol solution inverted the ζ-potentials of all siRNA lipoplexes from positive to negative. Notably, TC-1-12-based lipoplexes achieved strong gene silencing while minimizing interactions with erythrocytes. This study demonstrates the effectiveness of the two-step MEI method for preparing anionic siRNA lipoplexes.